Effects of Ca2+ channel antagonists on acetylcholine and catecholamine releases in the in vivo rat adrenal medulla

To elucidate the types of voltage-dependent Ca(2+) channels controlling ACh and catecholamine releases in the in vivo adrenal medulla, we implanted microdialysis probes in the left adrenal medulla of anesthetized rats and investigated the effects of Ca(2+) channel antagonists on ACh, norepinephrine, and epinephrine releases induced by nerve stimulation. The dialysis probes were perfused with Ringer solution containing a cholinesterase inhibitor, neostigmine. The left splanchnic nerves were electrically stimulated at 2 and 4 Hz before and after intravenous administration of Ca(2+) channel antagonists. omega-Conotoxin GVIA (an N-type Ca(2+) channel antagonist, 10 microg/kg) inhibited ACh release at 2 and 4 Hz by approximately 40%, norepinephrine release at 4 Hz by approximately 50%, and epinephrine release at 2 and 4 Hz by approximately 45%. A fivefold higher dose of omega-conotoxin GVIA (50 microg/kg) did not further inhibit these releases. omega-Conotoxin MVIIC (a P/Q-type Ca(2+) channel antagonist, 50 microg/kg) inhibited ACh and epinephrine releases at 4 Hz by approximately 30%. Combined omega-conotoxin GVIA (50 microg/kg) and MVIIC (250 microg/kg) inhibited ACh release at 2 and 4 Hz by approximately 70% and norepinephrine and epinephrine releases at 2 and 4 Hz by approximately 80%. Nifedipine (an L-type Ca(2+) channel antagonist, 300 and 900 microg/kg) did not change ACh release at 2 and 4 Hz; however, nifedipine (300 microg/kg) inhibited epinephrine release at 4 Hz by 20%, and nifedipine (900 microg/kg) inhibited norepinephrine and epinephrine releases at 4 Hz by 30%. In conclusion, both N- and P/Q-type Ca(2+) channels control ACh release on preganglionic splanchnic nerve endings while L-type Ca(2+) channels do not. L-type Ca(2+) channels are involved in norepinephrine and epinephrine releases on chromaffin cells.     

Vagosympathetic interactions in ischemia-induced myocardial norepinephrine and acetylcholine release

To elucidate the pathophysiological roles of vagosympathetic interactions in ischemia-induced myocardial norepinephrine (NE) and acetylcholine (ACh) release, we measured myocardial interstitial NE and ACh levels in response to a left anterior descending coronary occlusion in the following groups of anesthetized cats: intact autonomic innervation (INT, n = 7); vagotomy (VX, n = 6); local administration of atropine (Atro, n = 6); transection of the stellate ganglia (TSG, n = 5); local administration of phentolamine (Phen, n = 6); and combined vagotomy and transection of the stellate ganglia (VX+TSG, n = 5). The maximum NE release was enhanced in the VX group (141 +/- 30 nmol/l, means +/- SE, P < 0.05) compared with the INT group (61 +/- 12 nmol/l). Neither the Atro (50 +/- 24 nmol/l) nor VX+TSG groups (84 +/- 25 nmol/l) showed enhanced NE release. The maximum ACh release was unaltered in the TSG and Phen groups compared with the INT group (19 +/- 4, 18 +/- 4, and 13 +/- 3 nmol/l, respectively). These findings indicate that the cardiac vagal afferent but not efferent activity reduced the ischemia-induced myocardial NE release. In contrast, the cardiac sympathetic afferent and efferent activities played little role in the ischemia-induced myocardial ACh release.     

Hypothermia reduces ischemia- and stimulation-induced myocardial interstitial norepinephrine and acetylcholine releases.

Although hypothermia is one of the most powerful modulators that can reduce ischemic injury, the effects of hypothermia on the function of the cardiac autonomic nerves in vivo are not well understood. We examined the effects of hypothermia on the myocardial interstitial norepinephrine (NE) and ACh releases in response to acute myocardial ischemia and to efferent sympathetic or vagal nerve stimulation in anesthetized cats. We induced acute myocardial ischemia by coronary artery occlusion. Compared with normothermia (n = 8), hypothermia at 33 degrees C (n = 6) suppressed the ischemia-induced NE release [63 nM (SD 39) vs. 18 nM (SD 25), P < 0.01] and ACh release [11.6 nM (SD 7.6) vs. 2.4 nM (SD 1.3), P < 0.01] in the ischemic region. Under hypothermia, the coronary occlusion increased the ACh level from 0.67 nM (SD 0.44) to 6.0 nM (SD 6.0) (P < 0.05) and decreased the NE level from 0.63 nM (SD 0.19) to 0.40 nM (SD 0.25) (P < 0.05) in the nonischemic region. Hypothermia attenuated the nerve stimulation-induced NE release from 1.05 nM (SD 0.85) to 0.73 nM (SD 0.73) (P < 0.05, n = 6) and ACh release from 10.2 nM (SD 5.1) to 7.1 nM (SD 3.4) (P < 0.05, n = 5). In conclusion, hypothermia attenuated the ischemia-induced NE and ACh releases in the ischemic region. Moreover, hypothermia also attenuated the nerve stimulation-induced NE and ACh releases. The Bezold-Jarisch reflex evoked by the left anterior descending coronary artery occlusion, however, did not appear to be affected under hypothermia.     

Silver ions induce Ca2+ release from the SR in vitro by acting on the Ca2+ release channel and the Ca2+ pump.

Silver nitrate (AgNO3) is a sulfhydryl oxidizing agent that induces a biphasic Ca2+ release from isolated sarcoplasmic reticulum (SR) vesicles by presumably oxidizing critical sulfhydryl groups in the Ca2+ release channel (CRC), causing the channel to open. To further examine the effects of AgNO3 on the CRC and the Ca2+-ATPase, Ca2+ release was measured in muscle homogenates prepared from rat hindlimb muscle using indo 1. Cyclopiazonic acid (CPA) and ruthenium red (RR) were used to inhibit the Ca2+-ATPase and block the CRC, respectively, before inducing Ca2+ release with both AgNO3 and 4-chloro-m-cresol (4-CMC), a releasing agent specific for the CRC. With AgNO3 and CPA, the early rapid rate of release (phase 1) was increased (P < 0.05) by 42% (314 +/- 5 vs. 446 +/- 39 micromol x g protein(-1) x min(-1)), whereas the slower, more prolonged rate of release (phase 2) was decreased (P < 0.05) by 72% (267 +/- 39 vs. 74 +/- 7.7 micromol x g protein(-1) x min(-1)). RR, in combination with AgNO3, had no effect on phase 1 (P > 0.05) (314 +/- 51 vs. 334 +/- 43 micromol x g protein(-1) x min(-1)) and decreased phase 2 (P < 0.05) by 65% (245 +/- 34 vs. 105 +/- 8.2 micromol x g protein(-1) x min(-1)). With 4-CMC, CPA had no effect (P > 0.05) on either phase 1 or 2. With addition of RR, phase 1 was reduced (P < 0.05) by 59% (2,468 +/- 279 vs. 1,004 +/- 87 micromol x g protein(-1) x min(-1)), and RR completely blocked phase 2. Both AgNO3 and 4-CMC fully inhibited Ca2+-ATPase activity measured in homogenates. These findings indicate that AgNO3, but not 4-CMC, induces Ca2+ release by acting on both the CRC and the Ca2+-ATPase.     

The Ca2+ antagonists binding cytosolic protein has properties of the Ca2+ channel.

In our previous work (Krizanová et al. 1989) we have described a protein from rabbit skeletal muscle cytosolic fraction, which is able to bind dihydropyridines and phenothiazines. In the present work conclusive evidence is provided for the ability of the phospholipid-reconstituted cytosolic protein to transport calcium. The calcium transport was stimulated by BAY K 8644 and inhibited in the presence of PN 200-110. Our observations were confirmed also by electrophysiological measurements on planar lipid bilayers. The possibility that the cytosolic fraction was contaminated with membranes could be definitely ruled out. Nevertheless, the nature of the protein under study is still in the frame of guess.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. II. Releases involving a Ca2+-induced Ca2+ release channel

Calcium ions that have been preloaded into isolated sarcoplasmic reticulum subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances. We report here that not only caffeine, but also Ca2+ ions, thymol, quercetin, menthol, halothane, chloroform, 1-ethyl-2-methylbenzimidazole, ryanodine, tetraphenylboron, ketoconazole, miconazole, clotrimazole, W-7, doxorubicin, 5,5'-dithiobis-(2-nitrobenzoic acid), p-chloromercuribenzoic acid, and low concentrations of Ag+ induce Ca2+ release from such triadic sarcoplasmic reticulum. All these drugs induce increased undirectional Ca2+ efflux. We believe all these drug-induced Ca2+ releases are mediated by Ca2+ efflux through the same ion channel since these releases are all greatly attenuated when light sarcoplasmic reticulum is substituted for triads and are even more pronounced when transverse tubule-free terminal cisternae are substituted for triads, and all these forms of drug-induced Ca2+ release are inhibited by submicromolar concentrations of ruthenium red, and by submillimolar concentrations of tetracaine, 9-aminoacridine, and Ba2+, yet they are not affected by nifedipine even at a concentration of 50 microM.     

The voltage-gated Ca2+ channel is the Ca2+ sensor of fast neurotransmitter release

Previously it demonstrated that in the absence of Ca²⁺ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca²⁺] _i rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels 1:217–220.). These studies designate Ca²⁺ entry as opposed to [Ca²⁺] _i rise, essential for exocytosis. It led us to propose that the channel acts as the Ca²⁺ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein–protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J.; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J. 15:4100–4110; Wiser, O., Trus, M., Hernandez, A., Renström, E., Barg, S., Rorsman, P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A. 96:248–253). The kinetics of Ca_v1.2 (Lc-type) and Ca_v2.2 (N-type) Ca²⁺ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin I, a vesicle-associated protein, accelerated the activation kinetics indicating Ca_v2.2 coupling to the vesicle. The unique modifications of Ca_v1.2 and Ca_v2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Ca_v1.2 cytosolic domain Lc_753–893, acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca²⁺ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca²⁺-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca²⁺-dependent CAV (RRP) release, initiated by the binding of Ca²⁺ to the channel, upstream to intracellular Ca²⁺ sensor thus establishing the Ca²⁺ channel as the Ca²⁺ sensor of neurotransmitter release.     

Effects of Ca2+ agonist and antagonists on cytosolic free Ca2+ concentration: studies on Ca2+ channels in rat parotid cells

1. Effects of Ca2+ agonist and antagonists on cytosolic free Ca2+ concentration [( Ca2+]i)were studied using quin2. 2. Nicardipine (NIC), diltiazem (DIL) and verapamil (VER) had no effect on the rise in [Ca2+]i evoked by carbachol. Methoxamine-elevated [Ca2+]i was inhibited by VER but not by NIC and DIL. 3. All Ca2+ antagonists tested produced a decline of [Ca2+]i elevated by isoproterenol to the resting level. 4. The addition of 30 mM K+ gradually elevated [Ca2+]i in normal and Ca2+-free media, but it did not increase 45Ca2+ uptake into cells. BAY K 8644 did not increase [Ca2+]i. 5. We suggest that voltage-sensitive Ca2+ channels are lacking and that at least 2 distinct receptor-operated Ca2+ channels exist in rat parotid cells.     

Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes

This study examines whether fluid pressure (FP) modulates the L-type Ca²⁺ channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (16 dyn/cm²) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca²⁺ current (I_Ca) and cytosolic Ca²⁺ signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed I_Ca (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of I_Ca. The level of I_Ca suppression by FP depended on the level and duration of pressure. The Ba²⁺ current through the Ca²⁺ channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca²⁺ channel during FP stimulation. The cytosolic Ca²⁺ transients and the basal Ca²⁺ in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the I_Ca and on the Ca²⁺ transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca²⁺ buffer, eliminated the FP-induced acceleration of I_Ca inactivation and reduced the inhibitory effect of the FP on I_Ca by 80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca²⁺ release, eliminated the accelerating effect of FP on the I_Ca inactivation, and they reduced the inhibitory effect of FP on the I_Ca. These results suggest that the fluid pressure indirectly suppresses the Ca²⁺ channel by enhancing the Ca²⁺-induced intracellular Ca²⁺ release in rat ventricular myocytes. L-type Ca²⁺ current; fluid pressure; ventricular myocytes; cytosolic Ca²⁺ transient     

Ca2+ release through ryanodine receptors regulates skeletal muscle L-type Ca2+ channel expression

Skeletal muscle obtained from mice that lack the type 1 ryanodine receptor (RyR-1), termed dyspedic mice, exhibit a 2-fold reduction in the number of dihydropyridine binding sites (DHPRs) compared with skeletal muscle obtained from wild-type mice (Buck, E. D., Nguyen, H. T., Pessah, I. N., and Allen, P. D. (1997) J. Biol. Chem. 272, 7360-7367 and Fleig, A., Takeshima, H., and Penner, R. (1996) J. Physiol. (Lond.) 496, 339-345). To probe the role of RyR-1 in influencing L-type Ca(2+) channel (L-channel) expression, we have monitored functional L-channel expression in the sarcolemma using the whole-cell patch clamp technique in normal, dyspedic, and RyR-1-expressing dyspedic myotubes. Our results indicate that dyspedic myotubes exhibit a 45% reduction in maximum immobilization-resistant charge movement (Q(max)) and a 90% reduction in peak Ca(2+) current density. Calcium current density was significantly increased in dyspedic myotubes 3 days after injection of cDNA encoding either wild-type RyR-1 or E4032A, a mutant RyR-1 that is unable to restore robust voltage-activated release of Ca(2+) from the sarcoplasmic reticulum (SR) following expression in dyspedic myotubes (O'Brien, J. J., Allen, P. D., Beam, K., and Chen, S. R. W. (1999) Biophys. J. 76, A302 (abstr.)). The increase in L-current density 3 days after expression of either RyR-1 or E4032A occurred in the absence of a change in Q(max). However, Q(max) was increased 85% 6 days after injection of dyspedic myotubes with cDNA encoding the wild-type RyR-1 but not E4032A. Because normal and dyspedic myotubes exhibited a similar density of T-type Ca(2+) current (T-current), the presence of RyR-1 does not appear to cause a general overall increase in protein synthesis. Thus, long-term expression of L-channels in skeletal myotubes is promoted by Ca(2+) released through RyRs occurring either spontaneously or during excitation-contraction coupling.     

Effects of compound 48/80 on the Ca2+ release by reversal of the Ca2+ pump and by the Ca2+ channel of sarcoplasmic reticulum membranes

The effect of the calmodulin antagonist, compound 48/80, on the Ca2+ release from skeletal muscle sarcoplasmic reticulum was investigated. Both the Ca2+ release by reversal of the Ca2+ pump and the Ca2+ release by the Mg2(+)-controlled Ca2+ channel were studied. It was observed that, when reversal of the pump is inoperative and Mg2+ is not present in the reaction medium, 48/80 stimulates Ca2+ release from the vesicles. In contrast, in the presence of Mg2+, which blocks the Ca2+ channel, 48/80 inhibits Ca2+ release induced by ADP and Pi. This effect is strong at low concentrations of Pi (approximately 1 mM), whereas high concentrations (approximately 15 mM) protect the system against the drug. Furthermore, it was observed that 48/80 has a maximum effect on the channel-mediated Ca2+ release at concentrations of about 20 micrograms/ml, whereas maximal inhibition of the pump-mediated Ca2+ release occurs at concentrations of about 60-80 micrograms/ml. The results indicate that both the Ca2+ channel complex and the Ca2(+)-ATPase may be target systems for the effects of 48/80 on the Ca2+ transport activity of sarcoplasmic reticulum. However, the Ca2+ channel is more sensitive to the drug, suggesting an involvement of calmodulin on this mechanism of Ca2+ release.     

Role of intracellular Ca2+ in stimulation-induced increases in transmitter release at the frog neuromuscular junction

Under conditions of reduced quantal content, repetitive stimulation of a presynaptic nerve can result in a progressive increase in the amount of transmitter released by that nerve in response to stimulation. At the frog neuromuscular junction, this increase in release has been attributed to four different processes: first and second components of facilitation, augmentation, and potentiation (e.g., Zengel, J. E., and K. L. Magleby. 1982. Journal of General Physiology. 80:583-611). It has been suggested that an increased entry of Ca2+ or an accumulation of intraterminal Ca2+ may be responsible for one or more of these processes. To test this hypothesis, we have examined the role of intracellular Ca2+ in mediating changes in end-plate potential (EPP) amplitude during and after repetitive stimulation at the frog neuromuscular junction. We found that increasing the extracellular Ca2+ concentration or exposing the preparation to carbonyl cyanide m- chlorophenylhydrazone, ionomycin, or cyclopiazonic acid all led to a greater increase in EPP amplitude during conditioning trains of 10-200 impulses applied at a frequency of 20 impulses/s. These experimental manipulations, all of which have been shown to increase intracellular levels of Ca2+, appeared to act by increasing primarily the augmentation component of increased release. The results of this study are consistent with previous suggestions that the different components of increased release represent different mechanisms, and that Ca2+ may be acting at more than one site in the nerve terminal.     

Effects of alkaline pH on sarcoplasmic reticulum Ca2+ release and Ca2+ uptake.

Alkalinization-induced Ca2+ release from isolated frog or rabbit sarcoplasmic reticulum vesicles appears to consist of two distinct components: 1) a direct activation of ruthenium red-sensitive Ca2+ release channels in terminal cisternae and 2) an increased ruthenium red-insensitive Ca2+ efflux through some other efflux pathway distributed throughout the sarcoplasmic reticulum. The first of these releases exhibits an alkalinization-induced inactivation process and does not depend on the ruthenium red-insensitive form of Ca2+ release as a triggering agent for secondary Ca(2+)-induced Ca2+ release. Both releases are inhibited when the extravesicular (i.e. cytoplasmic) free [Ca2+] is reduced. This may reflect an increased sensitivity of the Ca2+ release channels to Ca2+ at alkaline pH. The pH sensitivity of the ruthenium red-sensitive Ca2+ release channels could be of significance during excitation-contraction coupling. The ruthenium red-insensitive form of Ca2+ release is less likely to be physiologically relevant, but it probably has contributed greatly to reports of alkalinization-induced decreases in net sarcoplasmic reticulum Ca2+ uptake, particularly under conditions where oxalate supported Ca2+ uptake is much less affected, as here.     

Osmoregulation of atrial myocytic ANP release: osmotransduction via cross-talk between L-type Ca2+ channel and SR Ca2+ release

Hyperosmolality has been known to increase ANP release. However, its physiological role in the regulation of atrial myocytic ANP release and the mechanism by which hyperosmolality increases ANP release are to be defined. The purpose of the present study was to define these questions. Experiments were performed in perfused beating rabbit atria. Hyperosmolality increased atrial ANP release, cAMP efflux, and atrial dynamics in a concentration-dependent manner. The osmolality threshold for the increase in ANP release was as low as 10 mosmol/kgH2O (approximately 3%) above the basal levels (1.55 +/- 1.71, 17.19 +/- 3.11, 23.15 +/- 5.49, 54.04 +/- 11.98, and 62.00 +/- 13.48% for 10, 20, 30, 60, and 100 mM mannitol, respectively; all P < 0.01). Blockade of sarcolemmal L-type Ca2+ channel activity, which increased ANP release, attenuated hyperosmolality-induced increases in ANP release (-13.58 +/- 4.68% vs. 62.00 +/- 13.48%, P < 0.001) and cAMP efflux but not atrial dynamics. Blockade of the Ca2+ release from the sarcoplasmic reticulum, which increased ANP release, attenuated hyperosmolality-induced increases in ANP release (13.44 +/- 7.47% vs. 62.00 +/- 13.48%, P < 0.01) and dynamics but not cAMP efflux. Blockades of Na+-K+-2Cl- cotransporter, Na+/H+ exchanger, and Na+/Ca2+ exchanger had no effect on hyperosmolality-induced increase in ANP release. The present study suggests that hyperosmolality regulates atrial myocytic ANP release and that the mechanism by which hyperosmolality activates ANP release is closely related to the cross-talk between the sarcolemmal L-type Ca2+ channel activity and sarcoplasmic reticulum Ca2+ release, possibly inactivation of the L-type Ca2+ channels.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Structure of Ca2+ release channel at 14 A resolution

The 14 A resolution structure of the 2.3 MDa Ca2+ release channel (also known as RyR1) was determined by electron cryomicroscopy and single particle reconstruction. This structure was produced using collected data used for our previous published structures at 22-30 A resolution, but now taking advantage of recent algorithmic improvements in the EMAN software suite. This improved map clearly exhibits more structural detail and allows better defined docking of computationally predicted structural domain folds. Using sequence-based fold recognition, the N-terminal region of RyR1, residues 216-572, was predicted to have significant structural similarity with the IP3-binding core region of the type 1 IP3R. This putative structure was computationally localized to the clamp-shaped region of RyR1, which has been implicated to have a regulatory role in the channel activity.     

Angiotensin II attenuates myocardial interstitial acetylcholine release in response to vagal stimulation

Although ANG II exerts a variety of effects on the cardiovascular system, its effects on the peripheral parasympathetic neurotransmission have only been evaluated by changes in heart rate (an effect on the sinus node). To elucidate the effect of ANG II on the parasympathetic neurotransmission in the left ventricle, we measured myocardial interstitial ACh release in response to vagal stimulation (1 ms, 10 V, 20 Hz) using cardiac microdialysis in anesthetized cats. In a control group (n = 6), vagal stimulation increased the ACh level from 0.85 +/- 0.03 to 10.7 +/- 1.0 (SE) nM. Intravenous administration of ANG II at 10 microg x kg(-1) x h(-1) suppressed the stimulation-induced ACh release to 7.5 +/- 0.6 nM (P < 0.01). In a group with pretreatment of intravenous ANG II receptor subtype 1 (AT(1) receptor) blocker losartan (10 mg/kg, n = 6), ANG II was unable to inhibit the stimulation-induced ACh release (8.6 +/- 1.5 vs. 8.4 +/- 1.7 nM). In contrast, in a group with local administration of losartan (10 mM, n = 6) through the dialysis probe, ANG II inhibited the stimulation-induced ACh release (8.0 +/- 0.8 vs. 5.8 +/- 1.0 nM, P < 0.05). In conclusion, intravenous ANG II significantly inhibited the parasympathetic neurotransmission through AT(1) receptors. The failure of local losartan administration to nullify the inhibitory effect of ANG II on the stimulation-induced ACh release indicates that the site of this inhibitory action is likely at parasympathetic ganglia rather than at postganglionic vagal nerve terminals.     

Effects of hemorrhage and opiate antagonists on adrenal release of neuropeptides in cats

Concurrent levels of methionine-enkephalin (ME), neuropeptide Y (NPY), peptide YY (PYY), neurotensin (NT), vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK) and bombesin (BMB) were measured in adrenal vein (AD), femoral vein (FV) and femoral artery (FA) under baseline conditions and during hypotensive hemorrhage (HTH) in halothane anesthetized cats (Group II, n = 6) and compared to a non-bled control group (Group I, n = 6). Five cats (Group III) received an IV bolus of naltrexone (1 mg/kg) followed by a continuous infusion prior to induction of HTH. A blood volume loss of approximately 40% evoked a selective increase in AD levels of ME, NPY, PYY and NT. No differences in regard to hemodynamics and pattern of neuropeptide levels were observed between Group II and Group III. Administration of naloxone (1 mg/kg, IV) in Group I and Group II at the end of the experiment led to a significant increase in MABP in both groups but did not evoke changes in neuropeptide levels. We conclude that adrenal neuropeptide release during hypotensive hemorrhage is not modulated by actions on opiate receptors in the halothane anesthetized cat.